Development of novel enkephalin analogues that have enhanced opioid activities at both μ and δ opioid receptors

Article abstract of DOI:10.1021/jm061465o

Enkephalin analogues with a 4-anilidopiperidine scaffold have been designed and synthesized to achieve therapeutic benefit for the treatment of pain due to mixed μ and δ opioid agonist activities. Ligand 16, in which a Dmt-substituted enkephalin-like stru

Full text of DOI:10.1021/jm061465o

5528
J. Med. Chem. 2007, 50, 5528-5532
Development of Novel Enkephalin Analogues that Have Enhanced Opioid Activities at Both µ
and δ Opioid Receptors
Yeon Sun Lee,† Ravil Petrov,† Chad K. Park,† Shou-wu Ma,‡ Peg Davis,‡ Josephine Lai,‡ Frank Porreca,‡
Ruben Vardanyan,† and Victor J. Hruby*,†
Departments of Chemistry and Pharmacology, UniVersity of Arizona, Tucson, Arizona 85721
ReceiVed December 21, 2006
Enkephalin analogues with a 4-anilidopiperidine scaffold have been designed and synthesized to achieve
therapeutic benefit for the treatment of pain due to mixed µ and δ opioid agonist activities. Ligand 16, in
which a Dmt-substituted enkephalin-like structure was linked to the N-phenyl-N-piperidin-4-yl propionamide
moiety, showed very high binding affinities (0.4 nM) at µ and δ receptors with an increased hydrophobicity
(aLogP ) 2.96). This novel lead compound was found to have very potent agonist activities in MVD (1.8
nM) and GPI (8.5 nM) assays.
Introduction
Neuropathic pain, which is associated with disease or injury
of the nervous system and characterized by the presence of
spontaneous ongoing types of pain, is particularly difficult to
treat.1,2 Morphine is a very potent µ opioid agonist and one of
the most commonly used drugs in the treatment of pain, even
though it has serious side effects such as constipation, tolerance,
and dependence for long-term usage.2,3 Recent studies have
shown that the tolerance to morphine can be reduced by co-
administration of a small amount of DPDPE,a a well-known δ
opioid receptor agonist, and that modulation of both µ and the
δ opioid receptors may be beneficial based on the pharmaco-
logical and functional interactions between them.4,5 We herein
set out to design and discover novel, nonselective bivalent opioid
Figure 1. Chemical structure of opioid analogues.
ligands with a potential therapeutic advantage by reducing
adverse side effects in the treatment of pain.
On the basis of these facts, we sought additional opportunities
to increase the potency at both opioid receptors and change the
overall physicochemical properties by modifying the C-terminus
of enkephalin. Previously, our group developed several fentanyl-
derived moieties that can be ligated with various kinds of amino
acids (or peptide pharmacophores) to create a new class of
opioid analogues.14,15 We found that the propionyl moiety along
with the phenethyl part of fentanyl plays an important role in
opioid receptor binding and activation. Among them a ligand
that carries a Tyr-D-Ala-Gly-Phe opioid message sequence
showed good opioid affinity and bioactivity, suggesting that a
novel class of analgesics can be further developed by this
approach. Pursuing enhanced bioactivity at both opioid recep-
tors, a series of enkephalin analogues were designed and
synthesized in which three different structural moieties of
fentanyl (1-3) were attached to the C-terminus with or without
a linker and tyrosine moiety was replaced by 2,6-dimethyl-
tyrosine (Dmt; Figure 2, Scheme 1). These analogues were
designed to have a mixed µ and δ agonist profile with a potential
for increased cell permeability due to the lipophilic character
of the 4-anilido piperidine moiety (Table 1).16,17
Enkephalins are highly flexible pentapeptides that can exist
in numerous conformations (Figure 1).6 The SAR study of
enkephalins has shown that (i) an additional aromatic group
strongly enhanced receptor affinity and (ii) the COOH group
at the C-terminus played an important role in δ opioid receptor
selectivity.7-10
Natural opioid peptides show in general poor bioavailability,
mainly due to their inability to penetrate the blood-brain barrier
and rapid degradation in vivo by several peptidases. To
overcome these problems, diverse strategies such as insertion
of unnatural amino acids, introduction of conformational
constraints, and cyclization of linear peptides have been
adopted.11-13 In several cases, peptide analogues or peptido-
mimetics have been found to possess much higher biological
activity than that expected on the basis of simple binding affinity
studies.
* To whom correspondence should be addressed. Phone: (520)-621-
6332. Fax: (520)-621-8407. E-mail: hruby@u.arizona.edu.
†Department of Chemistry.
‡Department of Pharmacology.
a Abbreviations: Boc, tert-butyloxycarbonyl; BOP, (benzotriazole-1-
yloxy)-tris(dimethylamino)-phosphonium hexafluorophosphate; CHO, Chi-
nese hamster ovary; DALEA, [D-Ala2, Leu5]enkephalin amide; DMF, N,N-
dimethylformamide; hDOR, human δ opioid receptor; DPDPE, c[D-Pen2,D-
Pen5]enkephalin; DAMGO, [D-Ala2,NMePhe4,Gly5-ol]enkephalin; Dmt, 2,6-
dimethyltyrosine;GPI,guineapigisolatedileum;HOBt,1-hydroxybenzotriazole;
rMOR, rat µ opioid receptor; MVD, mouse vas deferens; NMM, N-
methylmorpholine; RP-HPLC, reverse phase high performance liquid
chromatography; SAR, structure-activity relationship; TFA, trifluoroacetic
acid; TLC, thin layer chromatography.
Results and Discussion
There is evidence that nonselective µ/δ ligands with different
combinations of agonist and (or) antagonist activities at each
of the opioid receptors can act as potent analgesics.18,19 These
bifunctional ligands bind to more than one opioid receptor while
producing the desired physiological effects with enhanced
10.1021/jm061465o CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/10/2007

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5529
Table 1. Hydrophobicity (aLogP) of the Opioid Ligands16-17
N-piperidin-4-yl-propionamide to the enkephalin-like structures.
In this regard, we modified the enkephalin-like structure along
with the insertion of a linker (Figure 2).
Figure 2. Design of opioid ligands.
Scheme 1a
The designed ligands were prepared by stepwise (for 10-12
and 17) or fragment (for 13-16) solution-phase peptide
syntheses using NR-Boc chemistry, and the products were
purified by preparative RP-HPLC to afford >98% pure com-
pounds in 40-50% overall yields (Scheme 1). During the chain
elongation, the peptide intermediates were isolated by precipita-
tion from appropriate organic solvents, usually diethyl ether,
with high purity.
Opioid binding affinities of these ligands for the human δ
opioid receptor (hDOR) or the rat µ opioid receptor (rMOR)
were determined by radioligand competition analysis using [3H]-
DPDPE for the δ opioid receptor and [3H]DAMGO for the µ
opioid receptor in cell membrane preparations from transfected
cells that stably express the respective receptor type. Opioid
agonist efficacy was examined by monitoring [35S]GTP-γ-S
binding. For functional characterization of the ligands at the δ
and µ opioid receptors, classical assays were performed to
evaluate their opioid agonist activities in the guinea pig isolated
ileum (GPI) and mouse vas deferens (MVD). These results
generally were comparable with the results from the [35S]GTP-
γ-S binding assay.
The new opioid ligands showed a very broad range of
bioactivities at the δ and µ opioid receptors depending on their
respective structures (Table 2).20,21 Comparing with the tet-
rapeptide amide, most ligands increased their binding affinities
at the δ opioid receptor with the ligation of the fentanyl moieties.
The most distinct observation was that the parts of the fentanyl
moiety attached to the C-terminus of the enkephalin-like
structure played an important role in determining selectivity for
the opioid receptors. Derivatization of peptides at the N- or
C-terminus has frequently been carried out in attempts to
improve the activity, bioavailability, and physicochemical
properties of potential drug candidates. In our case, these
modifications seemed to have the greatest effect on opioid
selectivity as measured by in vitro binding and functional assays.
Ligand 10, in which 1-phenethyl-piperidin-4-ylamine is attached
to the C-terminus of the enkephalin showed µ opioid receptor
selectivity in the functional (δ/µ ) 2.4) and GTP-γ-S binding
(δ/µ ) 4.2) assays, whereas ligand 14 with a N-phenyl-N-
piperidin-4-yl propionamide moiety gave highly δ opioid
receptor selective binding affinity (Ki ) 0.69 nM, δ/µ ) 0.03)
and agonist activity (IC50 ) 24 nM, δ/µ ) 0.12) in the MVD.
This trend of selectivity for the δ and µ opioid receptors was
observed in the whole series of ligands except for 17, which
has a linker inserted between the two moieties. In the case of
ligand 17, its µ opioid receptor selectivity over the δ receptor
a Reagents and conditions: (a) stepwise coupling (BOP/HOBt/NMM,
DMF, rt, 2-4 h) and deprotection (TFA, 0 °C, 20 min); (b) NH2-NH2,
DMF, 1 day; (c) 1 N KOH, EtOH; (d) succinic anhydride, EtOAc, 1 day;
(e) BOP/HOBt/NMM, DMF, 4 h; (f) TFA, 0 °C, 20 min.
efficacy and without many of the undesirable side effects of
selective µ opioid receptor ligands.19 Rational design of opioid
ligands that have mixed agonist activities for both µ and δ opioid
receptors was accomplished in these studies by the combination
of enkephalin-like structures (e.g., H-Tyr-D-Ala-Gly-Phe-) and
parts of the fentanyl moiety {(1-phenethyl-piperidin-4-yl)-
phenyl-amine or 1-phenethyl-piperidin-4-ylamine}. Taking into
account our experience in the development of µ opioid selective
4-anilidopiperidine analogues, we also introduced the N-phenyl-

5530 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
Table 2. Bioactivities of the Opioid Ligands
Brief Articles
[35S]GTP-γ-S binding
hDORa
rMORa
IC50
[3H]DPDPE b
[3H]DAMGO c
hDORd
EC50 Emax
(nM)i (%)
ns ns ns
rMORd
EC50 Emax
(nM)i (%)
(nM)e
Kih
(nM)
Kih
(nM)
MVD
(δ)
GPI
(µ)
j
j
f,g
f,g
f
f
No
LogIC50
LogIC50
LogEC50
nsk
LogEC50
8
-4.70 ( 0.17 9400
-4.93 ( 0.20 5400
ns
ns
53
52
52
ns
63
32
50
43
1%
0%
10
11
12
13
14
15
16
17
-7.51 ( 0.12
-8.12 ( 0.11
-7.88 ( 0.12
14
3.7
6.1
-7.52 ( 0.26
-8.60 ( 0.08
-8.63 ( 0.16
14
1.2
1.1
-6.29 ( 0.15 510
-7.54 ( 0.09 29
-6.95 ( 0.10 110
92 -6.90 ( 0.31 125
78 -7.79 ( 0.09 16
32 -7.96 ( 0.14 11
380 ( 80 160 ( 50
250 ( 50 47 ( 12
290 ( 70 95 ( 6
-4.87 ( 0.23 7000
-4.96 ( 0.31 5700
ns
ns
ns ns
ns
3%
24 ( 2
10%
3%
200 ( 60
15%
-8.75 ( 0.08
-6.40 ( 0.19
-9.10 ( 0.10
-8.18 ( 0.08
0.69 -7.24 ( 0.24
23
40
-7.43 ( 0.39 37
72 -7.39 ( 0.41 41
15 -7.08 ( 0.29 82
24 -9.05 ( 0.18 0.88
71 -7.63 ( 0.25 24
-7.44 ( 0.19 37
69
180
-7.05 ( 0.07
-6.78 ( 0.20 170
0.36 -9.08 ( 0.22
0.38 -9.12 ( 0.17 0.77
1.8 ( 0.2 8.5 ( 3.3
250 ( 70 120 ( 50
3.2
-7.92 ( 0.06
-8.24 ( 0.13
5.7
-6.82 ( 0.20 150
DAMGO
DPDPE
150
8.80 ( 0.25 1.6
-6.72 ( 0.17 190
YDAGF-
NH2
-6.14 ( 0.16
300
2.4
2.8
7.7
44 -7.98 ( 0.22 13
99
120 ( 10 47 ( 10
DALEA20,21
7.6
8.3
a Competition analyses were carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the respective receptor
types. b Kd ) 0.50 ( 0.1 nM. c Kd ) 0.85 ( 0.2 nM. d Expressed from CHO cell. e Concentration at 50% inhibition of muscle contraction at electrically
stimulated isolated tissues. f Logarithmic values determined from the nonlinear regression analysis of data collected from at least two independent experiments.
g Competition against radiolabeled ligand. h Antilogarithmic value of the respective IC50. i Antilogarithmic value of the respective EC50. j Net total bound/
basal binding × 100 ( SEM. k ns: not saturated.
in GTP-γ-S binding (δ/µ ) 6.3) and functional assays (δ/µ )
2.0) is more likely to be caused by the linker, 3-hydrazinocar-
bonyl-propionyl, rather than by the N-phenyl-N-piperidin-4-yl
propionamide moiety. Compared to 10, ligands 11 and 12
showed an increased selectivity for the µ opioid receptor as
evidenced by their enhanced biological activities (Ki ) 1.2 nM,
EC50 ) 16 nM at rMOR; IC50 ) 47 nM at GPI for 11, Ki ) 1.1
nM, EC50 ) 11 nM at rMOR; IC50 ) 95 nM at GPI for 12).
There were subtle differences in the biological activities of 11
and 12. The additional aromatic group on the piperidin-4-
ylamine moiety in ligand 12 did not enhance biological activities
at either receptor. Ligand 11, which has a less-hindered and
more flexible structure at the C-terminus of the enkephalin, can
bind better to the opioid receptors. This is especially noted in
the µ opioid receptor.
Ligand 14 in which N-phenyl-N-piperidin-4-yl propionamide
was attached to the C-terminus of enkephalin showed highly
selective biological activities for the δ opioid receptor in binding
(Ki ) 0.69 nM) and functional (IC50 ) 24 nM at MVD) assays.
It is worthwhile to note that the moiety at the C-terminal
reversed the µ selectivity (δ/µ ) 107 and 2.5 in the binding
and functional assays, respectively) of tetrapeptide amide (H-
Tyr-D-Ala-Gly-Phe-NH2) to the δ selectivity (δ/µ ) 0.03 and
0.12 in the binding and functional assays, respectively) in ligand
14, and the insertion of the flexible linker inverted again its
selectivity in the GTP-γ-S binding and the functional assays
from favoring the δ opioid to the µ opioid receptor as evidenced
by the 4.1- to 10-fold decreased δ opioid and 1.6- to 4.0-fold
increased µ opioid biological activities in ligand 17. As described
earlier, the insertion of the flexible linker to ligand 10 amplified
its µ opioid receptor selectivity in 11 and 12 by increasing
biological activities at the µ receptor much more than at the δ
opioid receptor. This observation suggests that there is a distinct
topographical difference between the δ and µ opioid receptors,
despite the high biological activities observed. The more flexible
structure seems to better fit the µ opioid receptor binding pocket.
Thus, our modifications at the C-terminus seem to primarily
determine the selectivity between the δ opioid receptor and the
µ opioid receptor.
residues and the free amine group of the N-terminus tyrosine.
In ligands 13 and 15, phenylalanine was truncated to investigate
the possible role of the phenyl group on the N-phenyl-N-
piperidin-4-yl propionamide moiety as a replacement. In this
case, both ligands lost their agonist activities in the MVD and
GPI assays. Moderate binding affinities of ligand 15 (Ki ) 180
nM and 40 nM at hDOR and rMOR, respectively) at both
receptors are more likely to be caused by the Dmt substitution
to facilitate opioid receptor recognition.18,22 These results
demonstrated that phenylalanine is an important key residue,
and the piperidine-linked aromatic group of fentanyl moiety
cannot be used as its substitute. This may be due to the lack of
topographical similarity that cannot be maintained by the longer
distance and different orientation of the alternate aromatic group.
Ligand 14 was a lead compound showing good binding affinities
at both δ and µ receptors, good biological efficacies, and agonist
functions even though its substantial selectivity for the δ
receptor. Because it has been known that substitution of Dmt
for tyrosine in opioid peptides results in a more pronounced
increase in µ receptor affinity than in δ receptor affinity,22-24
further modification on the ligand was performed to balance
its bioactivities at both receptors by replacing the tyrosine with
Dmt. The replacement dramatically increased biological activi-
ties for both δ and µ opioid receptors, especially much more
for the µ receptor. In binding assay, 16 showed a 2-fold increase
in affinity at the δ receptor (Ki ) 0.36 nM) and a 60-fold
increase in affinity at the µ receptor (Ki ) 0.38 nM) as compared
to 14, thus resulting in balanced binding affinities at both
receptors. In addition, 16 was on the order of 13-fold more
potent than 14 in the MVD (IC50 ) 1.83 nM) and 24-fold in
the GPI (IC50 ) 8.51 nM) assays. These results demonstrate
that the Dmt substitution for tyrosine in opioid peptides results
in a higher increase in µ receptor affinity than in δ receptor
affinity. In summary, the well-known effect of Dmt substitution
for the tyrosine residue increased the biological activities
dramatically yielding the most potent opioid ligand 16 for both
δ and µ opioid receptors. Binding affinities and EC50 values
(0.77 nM and 0.88 nM at hDOR and rMOR, respectively) were
in the subnanomolar range. The best IC50 values also were
obtained for this analogue.
In general, for the opioid receptors, the key pharmacophore
elements in a peptide structure are the phenylalanine and tyrosine

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5531
were triturated with diethyl ether. The crude powders were purified
by preparative RP-HPLC (10-40% of acetonitrile within 20 min)
to give pure 10-12 and 17 as white powders in 70, 52, 48, and
55% yields, respectively. For analytical data, see Supporting
Information.
Ligands 13-16. These ligands were prepared by stepwise
synthesis using the same coupling and deprotection methods starting
from 3. The crude peptide ligands were purified by preparative RP-
HPLC (10-50% of acetonitrile within 20 min) to give pure 13,
14, 15, and 16 as white powders in overall 35-42%. For analytical
data, see Supporting Information.
Conclusion
Novel enkephalin analogues in which different parts of the
fentanyl moiety were attached at the C-terminus of the tet-
rapeptide structure (Tyr-D-Ala-Gly-Phe-) have been designed
and synthesized. The mixed µ and δ opioid agonist activities
of these ligands were sought to enhance their analgesic effects
while reducing the undesired side effects and increasing
bioavailability. These analogues showed a broad range of
biological activities depending on their respective structures.
While all the analogues containing the tetrapeptide structure
retained their potencies for both µ and δ opioid receptors, their
selectivities for the receptors were highly dependent on the
topographical structure of the C-terminus. It is likely that the
more flexible and longer length of ligands 11 and 12 possess a
better fit for the µ opioid receptor binding pocket than that for
the δ opioid receptor. Furthermore, a reverse trend has been
noticed for the more constrained structures of ligands 14 and
16. The best result in all three assays (binding, GTP-γ-S,
functional) occurred when Tyr was replaced with Dmt, and
N-phenyl-N-piperidin-4-yl propionamide was attached to the
C-terminus of the tetrapeptide structure as in ligand 16. This
ligand may have a greater potential to penetrate the blood-
brain barrier due to the lipophilic character of the N-phenyl-
N-piperidin-4-yl-propionamide moiety.
Radioligand Labeled Binding Assay, [35S]GTP-γ-S Binding
Assay, GPI and MVD in Vitro Bioassay. The methods were
carried out according to that previously described.25
Acknowledgment. The work was supported by grants from
the USDHS, National Institute on Drug Abuse (DA-12394 and
DA-06284). We thank Margie Colie for assistance with the
manuscript.
Supporting Information Available: 1H NMR, MS, HRMS,
TLC, HPLC, and purity data of the ligands 8-17. This material is
available free of charge via the Internet at http://pubs/acs/org.
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Boc-Tyr-d-Ala-Gly-Phe-OEt (4). TFA.H-D-Ala-Gly-Phe-OEt
was prepared as previously described.18 The tripeptide ester (1.13
g, 2.6 mmol) and NR-Boc-Tyr (0.79 g, 2.8 mmol) were dissolved
in DMF (10 mL) and cooled in an ice bath for 10 min. BOP (0.79
g, 2.8 mmol), HOBt (0.38 g, 2.8 mmol), and NMM (0.56 mL, 5.2
mmol) were added to the reaction mixture and stirred for 3 h at rt.
After checking for the disappearance of the starting amine by TLC,
the mixture was concentrated under reduced pressure, followed by
dilution with EtOAc (50 mL). The organic layer was washed with
5% NaHCO3 (3 × 50 mL), 5% citric acid (2 × 50 mL), brine (1
× 50 mL), and water, consecutively, and dried over anhydrous Na2-
SO4. After filtering, the solution was concentrated under reduced
pressure to give a solid. The residue was washed with diethylether
(2 × 50 mL) and dried in vacuo to afford 1.46 g (96% yield) of 4
as a white powder. MS m/z 607.2 [M + Na]+.
Boc-Tyr-D-Ala-Gly-Phe-NH-NH2 (5). Compound 4 (467 mg,
0.8 mmol) in 8 mL of EtOH was treated with 55% hydrazine (0.8
mL) for 1 day and solidified with water to give pure 5 as a white
power in 89% yield: analytical RP-HPLC tR 16.9 min, purity
>98%; MS m/z 571.0 [M + H]+.
Boc-Tyr-D-Ala-Gly-Phe-OH (6). Compound 4 (584 mg, 1
mmol) in 8 mL of MeOH was treated with 2 mL of 1 M KOH for
2 h at rt and neutralized with 2 mL of 1 N HCl. The mixture was
concentrated under reduced pressure and extracted with EtOAc.
After concentration, a white solid was formed to give pure 6 (422
mg) in 76% yield: analytical RP-HPLC tR 14.3 min, purity >99%;
MS m/z 556.9 [M + H]+.
N-(1-Phenethyl-piperidin-4-yl)-succinamic Acid (7), N-(1-
Phenethyl-piperidin-4-yl)-N-phenyl-succinamic Acid (8), 4-Oxo-
4-[4-(phenyl-propionyl-amino)-piperidin-1-yl]-butyric acid (9).
Compound 1 (or 2, 3;9 408 mg, 2 mmol) was dissolved in 10 mL
of EtOAc, and succinic anhydride (300 mg, 3 mmol) was added to
the mixture. The reaction mixture was stirred for 3 h (3 days for 2,
1 h for 3) at rt, and the formed solid was filtered and washed with
EtOAc to give 7 (or 8, 12) in quantitative yield. Compound 7 MS
m/z 305.3 [M + H]+, 8 MS m/z 381.2 [M + H]+, 12 MS m/z 333.1
[M + H]+.
Ligands 10-12 and 17. NR-Boc-protected intermediates were
prepared by the same coupling method described above and
deprotected by TFA at 0 °C for 20 min. The mixtures were
evaporated and coevaporated with toluene. The concentrated solids

5532 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22
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